Tuberculosis (TB) is an ancient infectious disease that has maintained its ability to claim human lives within the modern world population. This disease has especially flourished within human immunodeficiency virus- (HIV-) infected individuals, with the devastating consequence of becoming a leading cause of death within this vulnerable population. Effective control of TB is critically hindered by the lack of quality diagnostics, a protective vaccine, and chemotherapy regimens with potency for drug-resistant TB patients. One aspect of TB biology that has not been investigated for translational breakthroughs is the role of bacterial small molecules. There is growing evidence that secretion of small molecules, such as cyclic-di-nucleotides, by pathogenic bacteria may influence host signal transduction to mediate bacterial survival and virulence. The genome of Mycobacterium tuberculosis (M.tb.), the causative agent of TB, encodes tens of nucleotide and di-nucleotide cyclases, indicating an important role for such small molecules in the context of human TB. Furthermore, research in our laboratory has demonstrated for the first time secretion of cyclic-di-nucleotides by M.tb. These M.tb.-secreted small molecules therefore represent an untapped opportunity to both enrich our general knowledge of the biological interplay between M.tb. and its host, as well as to identify biological indicators of disease. We hypothesize that M.tb. secretes small molecules that modulate host cell signaling events, generating both small molecule and host response signatures that could be exploited as biomarkers of TB. In this proposal, we have carefully defined three specific aims designed to provide direct and relevant data for the evaluation of our hypothesis. Aim 1: Characterization of the role of M.tb.-secreted small molecules 3',5'-cyclic-di-guanylate (c-di- GMP), 3',5'-cyclic-di-adenylate (c-di-AMP), and mycobactin T in the context of a mammalian infection. Aim 2: Analysis of c-di-GMP, c-di-AMP and mycobactin T levels and the host response specific to each of these small molecules in both the granulomatous mouse and rabbit cavitary TB models. Aim 3: Definition of human TB biomarker signatures. Identification and characterization of such molecular signatures would serve as a significant and novel contribution to the TB research field, with the potential to greatly accelerate the development of new diagnostics and tools for the clinical analysis of new TB drugs and drug regimens.